The objectives of the proposed research are to investigate the pathway and control of long chain fatty acid oxidation. Our first plan is to purify carnitine palmitoyltransferases A and B from mitochondria. We will use affinity chromatography with carnitine coupled via various length spacers to Sepharose 4B. The purified transferases will be characterized. Antisera will be produced to the two purified enzymes. The orientation of the two carnitine palmitoyltransferases in the mitochondrial inner membrane will be studied using degradation by proteolytic enzymes, release by phospholipase A and neutralization with antiserum. We plan to evaluate the pathway from transferase A carnitine/acylcarnitine translocase transferase B in liver and then skeletal muscle. The difference in Km for carnitine with skeletal muslce carnitine palmitoyltransferase A will be explored further. The site and mechanism of known inhibitors of the "transferase system" will be studied. Malonyl-CoA, acyl-d-carnitines, 2-bromopalmitoyl-CoA and 2-tetradecylglycidic acid. Although these inhibitors have been previously studied there are major problems in defining their exact site of action. We plan to develop the necessary derivatization procedures to esterify and then separate for analytical measurements the acylcarnitines by pressure liquid chromatography. This direct method of measurement will be applied to both animal and human in vivo studies and in vitro studies using isolated hepatocytes, peroxisomes, and mitochondria. With this approach the data required to identify the specific role of acylcarnitines in altered metabolism can be obtained. We will examine acyl-CoA dehydrogenase as a possible rate-limiting step for mitochondria beta-oxidation. We will study the effect of extramitochondrial carnitine on intramitochondrial acyl-CoA and the rate of fatty acid oxidation. Exploration of the relationship between peroxisomal and mitochondrial oxidation of fatty acids will use long chain dicarboxylic acids as a substrate and use the isolated organelles as well as hepatocytes from control, riboflavin-deficient and clofibrate-treated rats. We hope to define factors involved in the control of long chain fatty acids oxidation.